Cardiac myocytes contain an extramyofibrillar cytoskeletal matrix responsible for linking myofibrils laterally to each other and to the plasma membrane. The composition of this cytoskeleton exhibits a striking homology to that of nucleated avian erythrocytes, being composed of a membrane-skeleton and an intermediate filament skeleton. In this proposal we propose to investigate the regulation of the expression and assembly of the cardiac membrane-skeleton and use it as a model system to analyze the assembly of the sarcolemma during cardiac morphogenesis. The membrane-skeleton of erythrocytes is composed of the transmembrane anion transporter, the cytoplasmic domain of which serves as a receptor for ankyrin, which in turn binds to the heterodimeric protein Alpha Beta spectrin. Spectrin binds in turn to actin, an interaction that is stabilized by a protein known as protein 4.1. We have discovered that analogues of all of these proteins are expressed in cardiac myocytes. We propose four classes of experiments. In the first we plan to analyze the structure and expression of the genes of these proteins during cardiac myocyte differentiation using cDNA probes for these molecules that we have isolated from chicken erythroid cells. With the exception of Alpha spectrin all the other cardiac membrane-skeleton polypeptides have different electrophoretic mobilities than their erythroid counterparts and in some instances so do their respective mRNAs. These experiments will determine the molecular basis of this tissue-specific divergence and the level at which their expression is regulated. Studies of the rates of synthesis, turnover and accumulation of each of these polypeptides during cardiac myocyte differentiation in tissue culture, will establish whether the accumulation of each of these polypeptides is regulated at the mRNA level or whether assembly is additionally regulated post-translationally. In vitro reconstitution and assembly studies onto erythrocyte or cardiac sarcolemma vesicles from components synthesized in a reticulocyte lysate will allow us to uncouple synthesis from turnover and from assembly and define in detail any post-translational events in the assembly of these proteins. Finally synthesis and assembly of the cardiac membrane-skeleton will be reconstituted and analyzed in vivo after microinjection of mRNA into Xenopus oocytes under conditions where the contribution of each component in the assembly process can be studied independently of the other components.